Startle suppression after mild traumatic brain injury is associated with an increase in pro-inflammatory cytokines, reactive gliosis and neuronal loss in the caudal pontine reticular nucleus
Introduction
Traumatic brain injury affects nearly 1.7 million Americans each year, and 75% of these injuries are classified as mild (mTBI). In addition to affecting cognition and mood, mTBI causes somatic symptoms such as headache, dizziness, fatigue and sleep disturbances, as well as sensorimotor dysregulation. Dysfunction of sensorimotor processes, such as simple reaction time and the acoustic startle reflex (ASR), may provide sensitive markers of mTBI (Barker-Collo et al., 2015).
The acoustic startle response (ASR) is a simple defensive response with a core neural circuitry located in the pons of the brainstem (Davis et al., 1982, Koch, 1999). Auditory information is relayed to the cochlear nucleus via the eighth cranial nerve (VIII). Information is transmitted from the cochlear nucleus to the caudal pontine reticular nucleus (PnC) before descending to the spinal cord via the reticulospinal tract and synapsing on spinal motor neurons. The PnC is especially important for the sensorimotor integration necessary for ASR (Lingenhohl and Friauf, 1994). The ASR neural circuit is well-conserved through mammalian species including humans (Pissiota et al., 2002) and ASR has been used in humans to investigate changes in emotion in anxiety disorders and after TBI (Saunders et al., 2006, Grillon et al., 1996).
Following mild TBI or moderate TBI, ASR is suppressed in animals and humans (Saunders et al., 2006, Lu, 2003, Washington et al., 2012, Wiley et al., 1996, Xing et al., 2013, Pang et al., 2015). While previous studies have observed suppressed ASR lasting 30 days after injury in rats (Wiley et al., 1996), humans demonstrate suppressed ASR lasting at least 1 year after TBI (Saunders et al., 2006). Despite the robust and long-lasting suppression of ASR following mild and moderate TBI, the neurobiological changes responsible for the suppression is still unknown.
Because of the importance of the PnC to ASR, the present study investigated changes in this region following mTBI. Besides its importance to ASR, the PnC is located nearby the locus coeruleus, a structure where p-tau and neurofibrillary tangles are observed early in Chronic Traumatic Encephalopathy (CTE) (Stein et al., 2014), a neurodegenerative disorder induced by sustaining multiple concussions over time. Long-lasting suppression of ASR following mTBI may reflect a general dysfunction of pontine nuclei including the locus coeruleus. Furthermore, the brainstem is the locus of key homeostatic and neuromodulatory systems, and the effect of mTBI is largely understudied in this brain region. Thus, understanding the mechanisms by which mTBI alters ASR may have implications for utilizing ASR as a behavioral marker of mTBI, as well as elucidating the progression of brain injury to neurodegeneration in CTE.
It is well established that moderate and severe TBI induces glial activation (Loane and Kumar, 2016). In rodent models, injury increases the pro-inflammatory cytokines, interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α), and elevated levels of these mediators result in both cellular dysfunction and neurodegeneration (Shaftel et al., 2008, Gosselin and Rivest, 2007). Increased IL-1β in the brain triggers a cascade in which cyclooxygenase-2 activation facilitates further production of inflammatory mediators such as eicosanoids (Bartfai et al., 2007). Additionally, elevations in TNF-α can activate apoptotic pathways thus promoting neurotoxicity (Longhi et al., 2013). In rodent models of moderate and severe TBI, IL-1β and TNF-α mRNA is detectable up to 6 h post-injury in regions proximal to the site of impact such as the cortex and hippocampus (Fan et al., 1995, Fan et al., 1996, Raghupathi et al., 1995); levels of TNF-α protein are elevated after moderate brain injury in the cortex up to 4 h post-injury (Knoblach et al., 1999). However, Perez-Polo et al. (Perez-Polo et al., 2013) demonstrated that similar effects are also evident in mTBI. In the cortex and hippocampus proximal to the injury site, IL-1β and TNF-α protein levels were increased up to 6 h post-injury, as were levels of reactive gliosis (both microglial and astrocytic). Similar studies using weight-drop models found either a delayed increase of IL-1β, IL-6 and TNF-α 4–6 days after injury with no change during the first 2 days (Holmin et al., 1997), or an elevation of IL-1β mRNA and proteins 1, 3 and 7 days after injury in the cortex (Lv et al., 2014). Therefore, more studies are required to understand the nature of inflammatory processes after mTBI.
The present study was conducted to determine whether glial activation in the PnC, a critical site for ASR, could account for the suppressed ASR following mTBI. On one hand, mTBI-induced glial activation is expected given the previous literature. On the other hand, the PnC is quite remote from the area of impact, and previous studies have reported the enhanced inflammatory responses in brain areas close to the impact zone.
Section snippets
Subjects
Male Sprague Dawley rats (approximately 3 months of age, 300–350 g at the start of the studies) were housed individually in a room with a 12:12 h light:dark cycle. Food and water were available ad libitum. All procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the IACUC of the Veterans Affairs Medical Center at East Orange, New Jersey.
Surgery
Rats were prepared for lateral fluid percussion injury, as previously described (Neuberger et al.,
Injury characteristics
Rats were divided into four groups for this study: mTBI rats euthanized at PID1 (n = 9), PID7 (n = 11), PID21 (n = 12) and SHAM (n = 14). SHAM rats were euthanized at PID1, PID7 and PID21, and data were pooled. Mean amplitudes of the fluid pressure pulse delivered to the three mTBI groups were as follows: 20.1 ± 0.9 psi for mTBI-PID1, 21.1 ± 0.7 psi for mTBI-PID7 and 20.8 ± 0.9 psi for mTBI-PID21. Comparison of apnea duration and latency of righting reflex is shown in Table 1. No significant difference in the
Discussion
The present study investigated the mechanism underlying a long-lasting suppression of ASR following mTBI. Following a mild lateral fluid percussion injury that resulted in no gross brain damage, a suppression of both sensitivity and amplitude of ASR was observed as long as 21 days after injury. These results were consistent with our and others previous reports (Xing et al., 2013, Pang et al., 2015). The focus of the present study was to investigate the mechanisms of the long-lasting suppression
Funding sources
The research presented in the current study was funded by the Biomedical Laboratory Research & Development Service of the Department of Veterans Affairs Office of Research & Development (grant I01BX000132), New Jersey Commission on Brain Injury Research (grant CBIR11PJT003), National Institutes of Health (grant RO1-NS44373), and the Stress and Motivated Behavior Institute.
Disclosures
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The opinions and conclusions presented are those of the authors and are not the official position of the U.S. Department of Veterans Affairs.
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